Background: Compared with titanium (Ti) and other metal implant materials, poly(ether-ether ketone) (PEEK) shows outstanding biomechanical properties. A number of studies have also reported attractive bioactivity for nano-TiO2 (n-TiO2).Methods: In this study, n-TiO2/PEEK nanocomposites were prepared, taking advantage of the unique properties of both PEEK polymer and n-TiO2. The in vitro and in vivo bioactivity of these nanocomposites was assessed against a PEEK polymer control. The effect of surface morphology or roughness on the bioactivity of the n-TiO2/PEEK nanocomposites was also studied. n-TiO2/PEEK was successfully fabricated and cut into disks for physical and chemical characterization and in vitro studies, and prepared as cylindrical implants for in vivo studies. Their presence on the surface and dispersion in the composites was observed and analyzed by scanning and transmission electron microscopy and X-ray photoelectron spectroscopy.Results: Bioactivity evaluation of the nanocomposites revealed that pseudopods of osteoblasts preferred to anchor at areas where n-TiO2 was present on the surface. In a cell attachment test, smooth PEEK showed the lowest optical density value (0.56 ± 0.07) while rough n-TiO2/PEEK exhibited the highest optical density value (1.21 ± 0.34, P < 0.05). In in vivo studies, the percent bone volume value of n-TiO2/PEEK was approximately twice as large as that of PEEK (P < 0.05). Vivid three-dimensional and histologic images of the newly generated bone on the implants further supported our test results.Conclusion: Our study demonstrates that n-TiO2 significantly improves the bioactivity of PEEK, especially if it has a rough composite surface. A n-TiO2/PEEK composite with a rough surface could be a novel alternative implant material for orthopedic and dental applications.